Phenotypic and genotypic assessment of fluoroquinolones and aminoglycosides resistances in Pseudomonas aeruginosa collected from Minia hospitals, Egypt during COVID-19 pandemic.

BACKGROUND: One of the most prevalent bacteria that cause nosocomial infections is Pseudomonas aeruginosa. Fluoroquinolones (FQ) and aminoglycosides are vital antipseudomonal drugs, but resistance is increasingly prevalent. The study sought to investigate the diverse mechanisms underlying FQ and aminoglycoside resistance in various P. aeruginosa strains particularly during the COVID-19 crisis. METHODS: From various clinical and environmental samples, 110 P. aeruginosa isolates were identified and their susceptibility to several antibiotic classes was evaluated. Molecular techniques were used to track target gene mutations, the presence of genes encoding for quinolone resistance, modifying enzymes for aminoglycosides and resistance methyltransferase (RMT). Efflux pump role was assessed phenotypically and genotypically. Random amplified polymorphic DNA (RAPD) analysis was used to measure clonal diversity. RESULTS: QnrS was the most frequently encountered quinolone resistance gene (37.5%) followed by qnrA (31.2%) and qnrD (25%). Among aminoglycoside resistant isolates, 94.1% harbored modifying enzymes genes, while RMT genes were found in 55.9% of isolates. The aac(6')-Ib and rmtB were the most prevalent genes (79.4% and 32.3%, respectively). Most FQ resistant isolates overexpressed mexA (87.5%). RAPD fingerprinting showed 63.2% polymorphism. CONCLUSIONS: Aminoglycosides and FQ resistance observed in this study was attributed to several mechanisms with the potential for cross-contamination existence so, strict infection control practices are crucial.

Phytotoxicity and genotoxicity study of reactive red 141 dye on mung bean (Vigna radiata (L.) Wilczek) seedlings.

BACKGROUND: Reactive Red (RR) 141 dye is widely used in various industrial applications, but its environmental impact remains a growing concern. In this study, the phytotoxic and genotoxic effects of RR 141 dye on mung bean seedlings (Vigna radiata (L.) Wilczek) were investigated, serving as a model for potential harm to plant systems. METHODS AND RESULTS: Short-term (14 days) and long-term (60 days) experiments in paddy soil pot culture exposed mung bean seedlings to RR 141 dye. The dye delayed germination and hindered growth, significantly reducing germination percentage and seedling vigor index (SVI) at concentrations of 50 and 100 ml/L. In short-term exposure, plumule and radical lengths dose-dependently decreased, while long-term exposure affected plant length and grain weight, leaving pod-related parameters unaffected. To evaluate genotoxicity, high annealing temperature-random amplified polymorphic DNA (HAT-RAPD) analysis was employed with five RAPD primers having 58-75% GC content. It detected polymorphic band patterns, generating 116 bands (433 to 2857 bp) in plant leaves exposed to the dye. Polymorphisms indicated the appearance/disappearance of DNA bands in both concentrations, with decreased genomic template stability (GTS) values suggesting DNA damage and mutation. CONCLUSION: These findings demonstrate that RR 141 dye has a significant impact on genomic template stability (GTS) and exhibits phytotoxic and genotoxic responses in mung bean seedlings. This research underscores the potential of RR 141 dye to act as a harmful agent within plant model systems, highlighting the need for further assessment of its environmental implications.

Genetic relatedness and diversity of Capillaria species infecting bayad (Bagrus bajad) in upper Egypt.

BACKGROUND: This study investigates the genetic characteristics of Capillaria isolates from the infected fish, Bagrus bajad, and their relation to human Capillaria philippinensis using Random Amplified Polymorphic DNA (RAPD-PCR) analysis. Fifteen fish Capillaria were isolated and compared to identified human C. philippinensis using six primers: M-are, M-1, G-7, G-11, G-15, and G-18. RESULTS: All six primers successfully amplified DNA, highlighting their efficacy in distinguishing between human and fish Capillaria isolates. The analysis revealed distinctive banding patterns between fish and human isolates, with variations in size and number of DNA fragments. Additionally, genetic similarity analysis showed intriguing patterns of relatedness, with certain pairs exhibiting high similarity percentages. Comparative assessment of RAPD polymorphism demonstrated consistent findings of 100% polymorphism across all primers. The Unweighted Pair Group Method with Arithmetic Mean Algorithm (UPGMA) evaluated the closest relationship between human and fish isolates. These results underscore the utility of RAPD analysis in delineating the genetic diversity among Capillaria isolates from different hosts. CONCLUSION: Overall, this study contributes to our understanding of the genetic variability and relatedness among Capillaria isolates, shedding light on their evolutionary dynamics and zoonotic potential.

Limited genetic variability and spatial population structure in grasshoppers between natural and metal-contaminated areas in Egypt.

Pollutants in an environment can have long-term implications for the species living there, resulting in local adaptations with implications for their genetic structure. Heavy metal pollutants infiltrate soils and groundwater, bioaccumulate in food webs, and negatively impact biota. In this study, we investigated the degree to which the genetic structure and variability of the slender green-winged grasshopper (Aiolopus thalassinus (Fabricius) (Orthoptera: Acrididae)) were impacted by heavy metal pollution and distance. We used the random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) method to examine the genetic variability of populations in 3 heavy metal-polluted and 3 unpolluted locations across varying geographical distances in Egypt. The heavy metal concentrations of cadmium, copper, lead, and zinc were measured from the grasshopper tissue and soils. Sixty-nine unique and polymorphic bands were produced by 4 primers. Cluster and principal component analyses separated the populations inside and outside Cairo into 2 main branches, which were further divided into smaller branches corresponding to their geographical regions. We found no differences in the Shannon genetic diversity index between populations or with increasing heavy metal concentrations in either the soil or the grasshopper tissue. Our results showed a greater genetic variation among populations than between populations within the same location, indicating populations within locations were less differentiated than those between locations. The moderate correlation between genetic similarity and spatial distance suggests geographical isolation influenced grasshopper population differentiation. Based on the RAPD analysis, environmental pollutants and geographical distances impact the A. thalassinus population structure, potentially restricting gene flow between sites even at small spatial scales.

Characterizations of Ganoderma species causing basal stem rot disease in coconut tree.

The basal stem rot disease incidence ranged from 0 to 5% in Karnataka India during the year 2019-20. Twenty pathogenic isolates of Ganoderma sp varied with cultural characteristics and virulence on coconut seedlings of the variety Tipatur Tall. The identity of each isolate was confirmed through morphological characters and through ITS sequencing. Two isolates viz., G4 and G5 were identified as Ganoderma applanatum and remaining all isolates were identified as G. lucidum. The genetic diversity analysis of Ganoderma isolates was done using ten Random Amplified Polymorphic DNA (RAPD) and fifteen Inter Simple Sequence Repeat (ISSR) primers. Among the ten RAPD primers, only eight primers recorded polymorphism (33.30-66.70%). The primer SBS-Q3 exhibited the highest polymorphism of 66.70%. In case of ISSR primers, all primers recorded polymorphism (33.30-60.00%). The primer UBC866 was the most polymorphic primer with 60.0% polymorphism. RAPD and ISSR markers were compared for their efficacy in assessing the genetic diversity by taking the band frequency, Shannon's index, polymorphic information content, resolving power, and mean resolving power into consideration, and it was concluded that ISSR was marker of choice over RAPD. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03872-w.

Capsular gene distribution and RAPD typing of Streptococcus agalactiae isolated from pregnant women.

Streptococcus agalactiae has different virulence factors, from which the capsule has the most significant role in the pathogenesis of this organism. We aimed to investigate the distribution of more prevalent capsular genes among different Random Amplified Polymorphic DNA (RAPD) types of S. agalactiae isolated from pregnant women. A total of 106 isolates were collected from 420 vaginal and rectal swabs obtained from pregnant women. The specimens were transferred using Todd Hewitt Broth and were cultured on a blood agar containing antibiotics. The S. agalactiae isolates were identified by the standard microbiological and biochemical tests. The genomic DNAs of S. agalactiae isolates were extracted using an extraction kit. Then, the PCR method was used to detection of the capsular genes. Moreover, The RAPD PCR was used to genotyping of the isolates. The colonization rate of the pregnant women was 25.23%, and there was a statistically significant correlation between the weeks of gestation and the probability of colonization (p-value < 0.05). Also, 31 (29.24%) and 18 (16.98%) pregnant women had a history of abortion and membrane rupture, respectively. In addition, 20 (18.86%), 32 (30.18%), 4 (3.77%), and 6 (5.66%) isolates carried genes encoding capsular types Ia, Ib, III, and V, respectively. None isolates had the type II capsular gene, and other 44 isolates were non-typeable. Nine clones (clusters) of S. agalactiae were observed in the present study with 70% similarity, and 53 different types were identified among the isolates. Except for capsular types III and V that belonged to clones 3, 5, 7, and 9, other capsular types were detected in different RAPD types. We found that the capsular types Ib and Ia were predominant among pregnant women in this area, indicating their significance for vaccine designation. Also, our isolates showed a lower genotypic diversity in RAPD typing. This may be due to the same sources of most isolates.

Development of SCAR Markers for Genetic Authentication of Metarhizium acridum.

In this study, molecular typing using Randomly Amplified Polymorphic DNA (RAPD-PCR) was conducted on 16 original isolates of Metarhizium acridum obtained from locusts (Schistocerca piceifrons ssp. piceifrons.) in Mexico (MX). The analysis included reference strains of the genus Metarhizium sourced from various geographical regions. The isolates were identified by phenotypic (macro and micromorphology) and genotypic methods (RAPD-PCR and Amplified Fragment Length Polymorphisms (AFLP), through a multidimensional analysis of principal coordinates (PCoA) and a minimum spanning network (MST). Subsequently, Sequences-Characterized Amplified Region (SCAR) markers were developed for the molecular detection of M. acridum, these markers were chosen from polymorphic patterns obtained with 14 primers via RAPD-PCR. Phenotypic and genotypic characterization identified the MX isolates as M. acridum. Of all the polymorphic patterns obtained, only OPA04 and OPA05 were chosen, which presented species-specific bands for M. acridum, and further utilized to create SCAR markers through cloning and sequencing of the specific bands. The specificity of these two markers was confirmed via Southern hybridization. The SCAR markers (Ma-160OPA-05 and Ma-151OPA-04) exhibit remarkable sensitivity, detecting down to less than 0.1 ng, as well as high specificity, as evidenced by their inability to cross-amplify or generate amplification with DNAs from other strains of Metarhizium (as Metarhizium anisopliae) or different genera of entomopathogenic fungi (Cordyceps fumosorosea and Akanthomyces lecanii). These SCAR markers yield readily detectable results, showcasing high reproducibility. They serve as a valuable tool, especially in field applications.

Molecular characterization of trypanocide-resistant strains derived from a single field isolate of Trypanosoma evansi.

Four strains (SB-PR, SB-RS, SB-RD, and SB-RM) of Trypanosoma evansi (T. evansi) were used in this study. SB-PR is known to be trypanocide-sensitive, while the others are trypanocide-resistant to suramin, diminazene diaceturate, and melarsomine hydrochloride, respectively. SB-RS, SB-RD, and SB-RM are derivatives of a single field isolate of SB-PR. Trypanocide resistance will not only increase costs and decrease production efficiency but will also affect effective treatment strategies. Therefore, studies on this topic are important to avoid inefficient production and ineffective treatment. This paper aims to presents a comparative molecular characterization of the trypanocide-resistant strains compared to the parent population. Comparative molecular characterization of these strains based on a protein profile analysis performed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), DNA fingerprinting of random amplified polymorphic DNA (RAPD), and the molecular characterization of expression-site-associated 6 (ESAG6), variant surface glycoprotein (VSG), and T. evansi adenosine transporter-1 (TevAT1) gene sequences. The results show three derived strains (SB-RS, SB-RD, and SB-RM) exhibit different banding patterns than SB-PR. According to the RAPD results, SB-RS and SB-RD are different strains with DNA fingerprint similarities of about 77.8 %, while the DNA fingerprint of SB-RM has a similarity of 44.4 % to SB-RS and SB-RD. No differences in VSG were found among the four strains; however, ESAG6 showed differences in both nucleotide and amino acid sequences, as well as in its secondary and 3D structure. In conclusion, all molecular analyses of the ESAG6 gene showed that SB-PR, SB-RS, SB-RD, and SB-RM are different strains. Furthermore, SB-PR, SB-RS, SB-RD, and SB-RM did not exhibit the TevAT1 gene, so the resistance mechanism was determined to be unrelated to that gene.

Molecular characterization and multifunctional evaluation of lactic acid bacteria isolated from traditional sourdough.

The primary objective of this study was to characterize lactic acid bacteria (LAB) strains derived from sourdough for possible utilization as functional starters to produce sourdough and various cereal-based fermented foods. A total of 350 autochthonous LAB strains were isolated from 65 Type I sourdough samples and characterized using six random amplified polymorphic DNA (RAPD) primers at intra- and interspecific levels. Species identification of selected strains representing distinct clusters from RAPD analysis was performed based on the 16S rRNA region. The LAB strains were identified as Companilactobacillus crustorum (n = 135), Levilactobacillus brevis (n = 125), Latilactobacillus curvatus (n = 40), Companilactobacillus paralimentarius (n = 32), and Lactiplantibacillus plantarum (n = 18). A total of 66 LAB strains were selected for technological characterization along with two commercial strains. The characterization involved acidity development, EPS production potential, leavening activity, and growth abilities under harsh conditions. Principle component analysis (PCA) identified 2 Lp. plantarum and 14 Lev. brevis strains as the most relevant technologically. Among them, Lp. plantarum L35.1 and Lev. brevis L37.1 were resistant to tetracycline. Evaluation of probiotic characteristics (survival in pH 2.5 and bile presence, auto aggregation capacity, hydrophobic activity, antioxidant activity, antimicrobial activity) by PCA identified four strains with relevance to Lactobacillus rhamnosus GG (LGG), which were further selected for in vitro digestion assays. Lactiplantibacillus plantarum L7.8, Lev. brevis L55.1, and L62.2 demonstrated similar viability indices to LGG, along with increased auto aggregation capacity and antioxidant activity. These strains are promising as candidate starters for producing sourdough and sourdough-related fermented food products.

Determination of antibiotic resistance patterns and genotypes of Escherichia coli isolated from wild birds.

BACKGROUND: Curbing the potential negative impact of antibiotic resistance, one of our era's growing global public health crises, requires regular monitoring of the resistance situations, including the reservoir of resistance genes. Wild birds, a possible bioindicator of antibiotic resistance, have been suggested to play a role in the dissemination of antibiotic-resistant bacteria. Therefore, this study was conducted with the objective of determining the phenotypic and genotypic antibiotic resistance profiles of 100 Escherichia coli isolates of gull and pigeon origin by using the Kirby-Bauer disk diffusion method and PCR. Furthermore, the genetic relationships of the isolates were determined by RAPD-PCR. RESULTS: Phenotypic antibiotic susceptibility testing revealed that 63% (63/100) and 29% (29/100) of E. coli isolates were resistant to at least one antibiotic and multidrug-resistant (MDR), respectively. With the exception of cephalothin, to which the E. coli isolates were 100% susceptible, tetracycline (52%), kanamycin (38%), streptomycin (37%), ampicillin (28%), chloramphenicol (21%), trimethoprim/sulfamethoxazole (19%), gentamicin (13%), enrofloxacin (12%) and ciprofloxacin (12%) resistances were detected at varying degrees. Among the investigated resistance genes, tet(B) (66%), tet(A) (63%), aphA1 (48%), sul3 (34%), sul2 (26%), strA/strB (24%) and sul1 (16%) were detected. Regarding the genetic diversity of the isolates, the RAPD-PCR-based dendrograms divided both pigeon and gull isolates into five different clusters based on a 70% similarity threshold. Dendrogram analysis revealed 47-100% similarities among pigeon-origin strains and 40-100% similarities among gull-origin E.coli strains. CONCLUSIONS: This study revealed that gulls and pigeons carry MDR E. coli isolates, which may pose a risk to animal and human health by contaminating the environment with their feces. However, a large-scale epidemiological study investigating the genetic relationship of the strains from a "one health" point of view is warranted to determine the possible transmission patterns of antibiotic-resistant bacteria between wild birds, the environment, humans, and other hosts. Video Abstract.

Isolation, identification, antibiotic resistance profile and molecular analysis of Ornithobacterium rhinotracheal isolates from turkeys.

BACKGROUND: Ornithobacterium rhinotracheal (ORT) infects numerous birds, particularly chickens and turkeys. ORT is an emerging bacterial pathogen of global concern in the poultry industry. As ORT is rapidly spreading throughout commercial poultry, it requires intensive studies of its epidemiology, diagnostic procedures, molecular typing, virulence genes and antimicrobial resistance. OBJECTIVES: The present study was conducted in isolation and identification of ORT from slaughtered turkeys. METHODS: Cleft palate swabs of 200 were collected from slaughtered turkeys and cultured on blood agar. ORT was characterized using biochemical tests and PCR targeting the ORT 16S rRNA gene. Virulence genes of isolates were determined targeting adenylate kinase (adk), copA and virulence-associated protein D (vapD) genes. Additionally, diversity of ORT isolates was performed by enterobacterial repetitive intergenic consensus (ERIC) and RAPD PCR. Disk diffusion was used to determine the antibiotic sensitivity of the isolates. RESULTS: ORT was identified in 23 (11.5%) samples using both the biochemical tests and PCR. The result of detecting virulence genes showed that all the isolates (23: 100%) had the adk gene, whereas two (8.7%) isolates had the copA gene, and seven (30.43%) isolates had the vapD gene. Molecular typing of isolates revealed 21 different patterns by RAPD PCR assay using M13 primer and 20 distinct patterns by ERIC PCR test. Both ERIC and RAPD PCR were distinctive methods for investigating the genetic diversity of ORT isolates. The antibiotic resistance test showed that 18 (78.26%) isolates were resistant to gentamicin, amikacin, cefazolin, streptomycin and penicillin. All isolates (100%) were resistant to cloxacillin and fosfomycin. CONCLUSIONS: This study showed the prevalence of ORT in turkey and high resistance of this bacterium to many common veterinary antibiotics. Moreover, both ERIC and RAPD PCR are distinctive methods for investigating the genetic diversity of ORT isolates. These data may help monitor antibiotic resistance and typing of ORT in epidemiological studies and serve as the foundation for designing region-specific vaccines for future use.

Identification of Sex-Associated Genetic Markers in Pistacia lentiscus var. chia for Early Male Detection.

Pistacia lentiscus var. chia is a valuable crop for its high-added-value mastic, a resin with proven pharmaceutical and cosmeceutical properties harvested from the male tree trunk. To achieve the maximum economic benefits from the cultivation of male mastic trees, it is important to develop early sex diagnosis molecular tools for distinguishing the sex type. Thus far, the work on sex identification has focused on Pistacia vera with promising results; however, the low transferability rates of these markers in P. lentiscus necessitates the development of species-specific sex-linked markers for P. lentiscus var. chia. To our knowledge, this is the first report regarding: (i) the development of species-specific novel transcriptome-based markers for P. lentiscus var. chia and their assessment on male, female and monoecious individuals using PCR-HRM analysis, thus, introducing a cost-effective method for sex identification with high accuracy that can be applied with minimum infrastructure, (ii) the effective sex identification in mastic tree using a combination of different sex-linked ISSR and SCAR markers with 100% accuracy, and (iii) the impact evaluation of sex type on the genetic diversity of different P. lentiscus var. chia cultivars. The results of this study are expected to provide species-specific markers for accurate sex identification that could contribute to the selection process of male mastic trees at an early stage for mass propagation systems and to facilitate future breeding efforts related to sex-linked productivity and quality of mastic resin.

Trueperella Pyogenes-Strain Diversity and Occurrence in Dairy Herds.

Trueperella (T.) pyogenes is a mastitis-causing pathogen formerly known to cause severe clinical mastitis (CM), especially during the summer, leading to milk losses and low recovery rates. Unfortunately, its transmission behavior within herds is unclear. The diversity and occurrence of T. pyogenes were monitored to gain an initial insight into the infection transmission behavior of T. pyogenes in dairy herds and to lay a foundation for following targeted investigations. CM milk samples were collected from German herds, and one Swedish farm was sampled for isolates from subclinical mastitis. All in all, 151 T. pyogenes isolates from 16 herds were isolated, identified by MALDI TOF analysis and typed with RAPD PCR. Of these, 17 isolates originated from subclinical mastitis cases. We found that T. pyogenes mastitis occurred year-round, and clinical mastitis cases were caused by multiple strains (31 affected animals/28 strains). Instances of multiple cows being infected with the same T. pyogenes strain were rare and typically only involved a small number of animals at a time. However, if several quarters of a cow were affected, it was likely the same strain. Unlike clinical infections, subclinical T. pyogenes infections, in one investigated farm, harbored a dominant strain. Additionally, we found that T. pyogenes infections tended to persist and stay within a herd for a minimum of 7 months in the same or different cows.

Evaluation of Primers OPF-01, P54, and 1253 to Identify A. fumigatus, A. flavus, and A. niger from Polymorphic Patterns Obtained by RAPD-PCR.

We evaluated the specificity of the primers OPF-01, P54, and 1253 to identify A. fumigatus, A. flavus, and A. niger, respectively, with the RAPD-PCR method. Eighty-two isolates belonging to the sections Fumigati, Flavi, and Nigri were used. The isolates were identified by phenotypic (macro- and micromorphology) and genotypic (partial sequences of the BenA gene) methods. The RAPD-PCR method was used to obtain polymorphic patterns with the primers OPF-01, P54, and 1253. The specificity of the polymorphic patterns of the isolates of each species was evaluated through the UPGMA clustering method and logistic regression model. All isolates of the genus Aspergillus were identified at the section level by macro- and micromorphology showing the typical morphology of the sections Fumigati, Flavi, and Nigri, and the species were identified by the construction of the phylogeny of the partial sequence of the BenA gene. The patterns' polymorphic strains obtained with the primers OPF-01, P54, and 1253 for the isolates of A. fumigatus, A. flavus, and A niger, respectively, showed the same polymorphic pattern as the reference strains for each species. To verify the specificity of the primers, they were tested with other species from the sections Fumigati, Flavi and Nigri. The results support that the primers OPF-01, P54, and 1253 generate polymorphic patterns by RAPD-PCR species specific to A. fumigatus, A. flavus, and A. niger, respectively.

Molecular characterization of multidrug resistant (MDR) clinical isolates of Pseudomonas aeruginosa from Nsukka, South Eastern Nigeria.

Background: P. aeruginosa is an important nosocomial pathogen with increasing resistance to antibiotics. Objective: This study was performed to evaluate the genetic relatedness of MDR clinical isolates of P. aeruginosa. Method: A total of 1000 samples were analysed in the study. Antibiotic resistance profiles of the isolates were determined using Kirby Bauer disk diffusion method. Polymerase chain reaction (PCR) and sequencing were simultaneously used to detect the consensus region of 16S rRNA. Genetic relatedness of the isolates was determined using restriction patterns from ALU 1 digest and random amplified polymorphic DNA. Results: Out of the 192 P. aeruginosa isolates recovered, 136 (78.83%) were multidrug resistant. Sequence analysis of the confirmed isolates (80.68%) revealed that all the isolates shared homology with each other and also showed sequence similarity to known strains of P. aeruginosa (ATCC 27853; KT 315654; KU 321274 and KT894767). The PCR-Restriction fragment length polymorphism (PCR-RFLP) analysis revealed that there was a lot of genetic relatedness among the isolates. The RFLP finger printing technique detected seven distinct RFLP types among the isolates. Conclusions: Thus, study shows that there is high prevalence of MDRPA and high degree of genetic relatedness among the MDRPA isolates circulating in Nsukka area.

Genomic characterization of Puccinia triticina using molecular marker technology / Caracterização genômica de Puccinia triticina usando tecnologia de marcadores moleculares

Leaf rust, caused by Puccinia triticina, is the most common rust disease of wheat. The fungus is an obligate parasite capable of producing infectious urediniospores. To study the genetic structure of the leaf rust population 20 RAPD primers were evaluated on 15 isolates samples collected in Pakistan. A total of 105 RAPD fragments were amplified with an average of 7 fragments per primer. The number of amplified fragments varied from 1 to 12. GL Decamer L-07 and GL Decamer L-01 amplified the highest number of bands (twelve) and primer GL Decamer A-03 amplified the lowest number of bands i.e one. Results showed that almost all investigated isolates were genetically different that confirms high genetic diversity within the leaf rust population. Rust spores can follow the migration pattern in short and long distances to neighbor areas. Results indicated that the greatest variability was revealed by 74.9% of genetic differentiation within leaf rust populations. These results suggested that each population was not completely identical and high gene flow has occurred among the leaf rust population of different areas. The highest differentiation and genetic distance among the Pakistani leaf rust populations were detected between the leaf rust population in NARC isolate (NARC-4) and AARI-11and the highest similarity was observed between NARC isolates (NARC-4) and (NARC-5). The present study showed the leaf rust population in Pakistan is highly dynamic and variable.

A ferrugem da folha, causada por Puccinia triticina, é a ferrugem mais comum do trigo. O fungo é um parasita obrigatório, capaz de produzir urediniósporos infecciosos. Para estudar a estrutura genética da população de ferrugem da folha, 20 primers RAPD foram avaliados em 15 amostras de isolados coletadas no Paquistão. Um total de 105 fragmentos RAPD foram amplificados com uma média de 7 fragmentos por primer. O número de fragmentos amplificados variou de 1 a 12. GL Decamer L-07 e GL Decamer L-01 amplificaram o maior número de bandas (doze), e o primer GL Decamer A-03 amplificou o menor número de bandas, ou seja, um. Os resultados mostraram que quase todos os isolados investigados eram geneticamente diferentes, o que confirma a alta diversidade genética na população de ferrugem da folha. Os esporos de ferrugem podem seguir o padrão de migração em distâncias curtas e longas para áreas vizinhas. Os resultados indicaram que a maior variabilidade foi revelada por 74,9% da diferenciação genética nas populações de ferrugem. Esses resultados sugeriram que cada população não era completamente idêntica e um alto fluxo gênico ocorreu entre a população de ferrugem da folha de diferentes áreas. A maior diferenciação e distância genética entre as populações de ferrugem da folha do Paquistão foram detectadas entre a população de ferrugem da folha no isolado NARC (NARC-4) e AARI-11 e a maior similaridade foi observada entre os isolados NARC (NARC-4) e (NARC-5). O presente estudo mostrou que a população de ferrugem da folha no Paquistão é altamente dinâmica e variável.

Genetic variations among the isolates of Bipolaris Maydis based on phenotypic and molecular markers

Abstract Maydis leaf blight, caused by Bipolaris maydis, is an important disease of maize crop in Khyber Pakhtunkhwa (KP) Pakistan. Fifteen isolates of the pathogen, collected across KP, were studied for variability based on phenotypic and molecular markers. Significant variability among the isolates was observed when assessed using phenotypic traits such as radial growth, spore concentration, fungicide sensitivity and virulence. The isolates were classified into six culture groups based on colour, texture and margins of the colony. Conidial morphology was also variable. These were either straight or slightly curved and light to dark brown in colour. Fungicide test showed significant variation in the degree of sensitivity against Carbendazim. Isolate Bm8 exhibited maximum radial growth on carbendazim spiked plates. Conversely, isolate Bm15 showed the lowest radial growth. Variations in virulence pattern of the isolates were evident when a susceptible maize variety Azam was inoculated with spores of B. maydis. Genetic variability amongst the isolates was also estimated by RAPD as well as sequencing of ITS region. The RAPD dendrogram grouped all the isolates into two major clusters. Average genetic distance ranged from 0.6% to 100%, indicating a diverse genetic gap among the isolates. Maximum genetic distance was found between isolates Bm9 and Bm10 as well as Bm2 and Bm8. Conversely, isolates Bm13 and Bm15 were at minimum genetic distance. Phylogenetic dendrogram based on sequencing of ITS region grouped all the isolates into a single major cluster. The clusters in both the dendrogram neither correlate to the geographical distribution nor to the morphological characteristics.

Resumo A ferrugem das folhas de maydis, causada por Bipolaris maydis, é uma doença importante da cultura do milho em Khyber Pakhtunkhwa (KP), Paquistão. Quinze isolados do patógeno, coletados em KP, foram estudados quanto à variabilidade com base em marcadores fenotípicos e moleculares. Variabilidade significativa entre os isolados foi observada quando avaliada por meio de características fenotípicas, como crescimento radial, concentração de esporos, sensibilidade a fungicida e virulência. Os isolados foram classificados em seis grupos de cultura com base na cor, textura e margens da colônia. A morfologia dos conídios também foi variável. Estes eram retos ou ligeiramente curvos e de cor marrom-claro a escuro. O teste de fungicida mostrou variação significativa no grau de sensibilidade ao carbendazim. O isolado Bm8 exibiu crescimento radial máximo em placas com adição de carbendazim. Por outro lado, o isolado Bm15 apresentou o menor crescimento radial. As variações no padrão de virulência dos isolados foram evidentes quando uma variedade de milho suscetível Azam foi inoculada com esporos de B. maydis. A variabilidade genética entre os isolados também foi estimada por RAPD, bem como sequenciamento da região ITS. O dendrograma RAPD agrupou todos os isolados em dois grupos principais. A distância genética média variou de 0,6% a 100%, indicando uma lacuna genética diversa entre os isolados. A distância genética máxima foi encontrada entre os isolados Bm9 e Bm10 e também entre Bm2 e Bm8. Por outro lado, os isolados Bm13 e Bm15 estavam a uma distância genética mínima. O dendrograma filogenético baseado no sequenciamento da região ITS agrupou todos os isolados em um único aglomerado principal. Os agrupamentos em ambos os dendrogramas não se correlacionam com a distribuição geográfica nem com as características morfológicas.

Tinea corporis intrafamilial infection in pets due to Microsporum canis

ABSTRACT Microsporum canis, one of the most widespread dermatophytes worldwide, is a zoonotic microorganism that transmits infection from reservoirs such as cats and dogs to humans. This microorganism is associated with Tinea corporis and other clinical manifestations; however, few studies have used genetic surveillance to determine and characterize the process of zoonotic transmission. In this study, we show a clear example of zoonotic transmission from a cat to an intrafamilial environment, where it caused Tinea corporis by infection with M. canis. Molecular characterization using the b-tubulin gene and Random Amplified Polymorphic DNA analysis made it possible to determine that the six isolates of M. canis obtained in this study belonged to the same genetic variant or clone responsible for reservoir-reservoir or reservoir-human transmission.

Detección clonal de cepas parentales de streptococcus agalactiae Lehmann y Neumann por amplificación aleatoria de ADN polimórfico / Clonal detection of streptococcus agalactiae Lehmann and Neumann parental strains by random amplification of polymorphic DNA

RESUMEN Streptococcus agalactiae (SGB) produce infecciones invasivas en neonatos siendo la transmisión materna la más frecuente. Estudios epidemiológicos utilizan técnicas moleculares que evalúan la diversidad genética, entre ellas la de amplificación aleatoria de ADN polimórfico (RAPD) que resulta ser accesible, sensible y utiliza cebadores arbitrarios para amplificar segmentos polimórficos de ADN mediante PCR. El objetivo fue determinar la relación clonal entre aislamientos de SGB recuperados de madres y sus respectivos recién nacidos. Se estudiaron por RAPD cuatro parejas de aislamientos de SGB obtenidos de hisopados vagino-rectales de madres y de hemocultivos de sus neonatos. Se utilizaron los cebadores OPS11, OPB17 y OPB18 para seleccionar uno con capacidad de discriminar entre cepas no relacionadas genéticamente. Se utilizó la fórmula de Hunter-Gaston que establece el índice de discriminación (D), cuando D>0,90 se considera que los aislamientos pertenecen a clones diferentes. Los perfiles de amplificación para los ocho aislamientos, empleando independientemente cada cebador, permitieron calcular un D=1 para OPS11, y D=0,84 para OPB17 y OPB18. Por lo tanto, OPS11 fue seleccionado para el estudio de la relación clonal de los aislamientos, encontrándose perfiles de amplificación similares por RAPD para cada par de cepas madre-recién nacido. Se observaron diferentes perfiles de amplificación entre los pares de cepas madre-recién nacido, lo que revela la discriminación entre cepas no relacionadas, resultados confirmados por electroforesis en campo pulsante (PFGE). Estos resultados indican transmisión vertical para cada caso estudiado y robustez del cebador OPS11. Se encontraron condiciones apropiadas del ensayo de RAPD, lo que es útil para estudios epidemiológicos.

ABSTRACT Streptococcus agalactiae (GBS) causes invasive infections in newborns, being the most frequent the maternal transmission. Epidemiological studies use molecular techniques that assess genetic diversity, including random amplification of polymorphic DNA (RAPD) that is found to be accessible, sensitive and uses arbitrary primers to amplify polymorphic segments of DNA by PCR. The objective was to determine the clonal relationship between GBS strains recovered from mothers and their respective newborns. Four pairs of GBS isolates obtained from vaginal-rectal swabs of mothers and blood cultures of their newborns were studied with RAPD. Primers OPS11, OPB17 and OPB18 were used to select one with the ability to discriminate between non-genetically related strains. The Hunter-Gaston formula that establishes the discrimination index (D) was used; when D>0.90, it is considered that the isolates belong to different clones. The amplification profiles for the eight isolates, using each primer independently, allowed to calculate a D=1 for OPS11, and D=0.84 for OPB17 and OPB18. Therefore, OPS11 was selected for the study of the clonal relationship of the isolates, and similar amplification profiles were found by RAPD for each mother-newborn pair of GBS isolates. Different amplification profiles were observed between pairs of mother-newborn strains, which reveals the discrimination between unrelated strains, confirmed by pulsating field electrophoresis (PFGE). These results indicated vertical transmission for each studied case and robustness of the OPS11 primer. Appropriate conditions of the RAPD trial were found, which is useful for epidemiological studies.

Evaluation of eight protocols for genomic DNA extraction of Hypostomus commersoni Valenciennes, 1836 (Loricariidae: Siluriformes) / Avaliação de oito protocolos na extração de DNA genômico de Hypostomus commersoni Valenciennes, 1836 (Loricariidae: Siluriformes)

Abstract The principle and the techniques applied in DNA extraction play a pivotal role in the obtention of a purified genetic material. The present study investigates the efficiency of eight protocols in the DNA extraction of Hypostomus commersoni, an essential component of South American freshwater ichthyofauna. The quality of samples was assessed through spectrophotometry, gel electrophoresis, and PCR-RAPD markers amplification. The efficiency of DNA extraction was influenced both by the method applied and the target-tissue of choice. Higher concentrations and yield of DNA were obtained from ocular tissue, with a positive spectrum of incubation in lysis buffer for up to 36 hours after sample collection, using fresh tissues and in the presence of a high concentration of Proteinase K (20 mg.ml-1). In these conditions, samples were successfully amplified. To date, there is no record of description for the parameters analyzed in this work, neither the description of RAPD markers for the species H. commersoni.

Resumo Os princípios e as técnicas aplicadas na extração de DNA desempenham um papel crucial na obtenção de material genético purificado. O presente estudo investiga a eficiência de oito protocolos na extração de DNA de Hypostomus commersoni, um importante componente ictiofaunístico de riachos da América do Sul. A qualidade das amostras foi avaliada por espectrofotometria, eletroforese em gel e amplificação por marcadores de PCR-RAPD. A eficiência da extração de DNA foi influenciada tanto pelo método aplicado quanto pelo tecido-alvo de escolha. Maiores concentrações e rendimento de DNA foram obtidos a partir do tecido ocular, com um espectro positivo de incubação em tampão de lise por até 36 horas após a coleta da amostra, utilizando tecidos frescos e na presença de alta concentração de proteinase K (20 mg.ml-1). Nestas condições, as amostras foram amplificadas com sucesso. Até o momento, não há registro de descrição para os parâmetros analisados neste trabalho, nem a descrição de marcadores RAPD para a espécie H. commersoni.